N5-(L-1-carboxyethyl)-L-ornithine synthase (CEO synthase, EC 1.5.1.-) from Lactococcus lactis K1 is composed of four identical subunits (MW = 35,500) and catalyzes the reductive condensation between pyruvic acid and delta-amino group of L-ornithine mediated by NADPH. Our goal is to investigate physico-chemical properties, stability towards heat and chemical denaturation, and to correlate structural changes in CEO synthase with individual events on the unfolding/refolding pathway. Enzyme activity measurements, UV and fluorescence spectroscopy, circular dichroism, light scattering, HPLC, gel-electrophoresis, and analytical ultracentrifugation are being used. The fluorescence of native CEO synthase showed distinct emissions from Tyr (19 Tyr/monomer) and the single Trp whereas after denaturation, Tyr fluorescence dominated the signal. While ornithine had no effect on the intrinsic protein fluorescence, pyruvate produced a small decrease in Trp fluorescence; in contrast, NADPH addition completely quenched Trp and only slightly decreased Tyr emission. Two classes of Tyr residues in the native protein were identified by pH titrations: 2 Tyr with pK approximately 8.6 (accounting for approximately 40 % of the intrinsic Tyr fluorescence) and 17 with pK approximately 11.3. CEO synthase was irreversibly inactivated by guanidine/HCl (approximately 0.4 M) and urea (approximately 2 M) at pH 7. Some salts (e.g., KCl) in the assay mixture as well as low concentrations of denaturants reversibly stimulated enzyme activity and partially protected the enzyme from inactivation by higher denaturant concentrations. The protein was insoluble in very low concentrations of GdnHCl, and the loss of secondary structure occurred between 2 and 4 M GdnHCl under which conditions the protein is fully soluble. Thermal unfolding of the tetrameric CEO synthase in the absence of denaturants was accompanied by precipitation at approximately 60 degreesC. Preincubation with substrates (L-ornithine, pyruvate, or NADPH) separately or combined resulted in a modest (approximately 1-3 degreesC) stabilization of the protein but did not eliminate aggregation.